This program has been concerned with study of the structure, function and interaction of the enzyme complexes of the mitochondrial electron transport/oxidative phosphorylation system, and with the mechanism of ATP synthesis. The research proposed for the next grant period includes the following: Complex I (NADH:ubiquinone oxidoreductase). The kinetic sequence of the electron carriers (FMN, 6-8 iron-sulfur (FeS) clusters) and the structure of complex I (>40 unlike subunits) arc not known. It is proposed to study the sequence of electron carriers by differential inactivation of FeS clusters, using iron chelators, mercurials, peroxynitrite, etc., or the proteins that house them, using proteases. Lesions in electron transfer will be investigated by enzymatic assays and electron paramagnetic resonance spectroscopy of modified samples +/- NAD(P)H +/- dithionite. Structure studies will involve cross-linking of complex I and simplified versions thereof to identify by immunoblotting the near-neighbor relationships particularly of redox carrier proteins, ubiquinone (Q) and inhibitor binding proteins, and mtDNA-encoded subunits. Complex III (ubiquinol-cytochrome c oxidoreductase). (a) Post steady-state oxidation of cyt. bL through the leak in the antimycin block occurs 10 times faster than that of bH. Also, ascorbate +/- TMPD reduction of bH (or bH + bL in energized submitochondrial particles, SMP) via cyt. c and Rieske FeS protein is inhibited not only by myxothiazol alone, but also by antimycin alone. These results do not agree with the Q-cycle or with the assigned inhibition site of antimycin. It is proposed to study electron transfer from ascorbate +/- TMPD to cyt. b in bovine SMP, Q-extracted bovine SMP and complex III, yeast SMP, and Q-deficient yeast SMP +/- various inhibitors. (b)Antimycin or reduction stabilizes complex III against chaotropic resolution. It is proposed to investigate these structural changes by cross-linking experiments to identify altered subunit proximities and by the effect of proteases to characterize the possible altered protease sensitivities of subunits. Structural alterations of complex III by antimycin may shed light on the mechanism of antimycin inhibition and on the enzyme's mechanisms of electron transfer and energy coupling. Complex V (ATP synthase). Energy communication from the membrane sector F0 to the catalytic sector F1 appears to involve conformational coupling, which is a difficult problem to investigate. OSCP is a subunit that connects F1 to F0 and can be reversibly removed from SMP. It is proposed to label OSCP at its single nonessential cysteine or several OSCP mutants containing displaced cysteine with (a) fluorescent maleimides, and (b) photoactivable heterobifunctional maleimides. Then reconstitute and investigate the effects of F1 ligands, F0 modifiers and SMP energization on the fluorescence of the probe in (a) and on the cross- linking pattern after photoactivation in (b). In addition, the effects of F1 ligands and SMP energization will be studied on the fluorescence of two F0 modifiers that bind at nonoverlapping sites. Mitochondrial defects cause a variety of debilitating human diseases, whose prevention and treatment require a knowledge of the structure and function of mitochondria at the molecular level.